Proteins increasing pancreatic beta cell number and methods of use

ABSTRACT

Disclosed herein are bacterial proteins that increase pancreatic beta (β) cell number and/or proliferation, methods of increasing β cell number and/or proliferation using such proteins, and methods of treating or inhibiting diabetes in a subject by administering such proteins to the subject. In some embodiments, the protein has at least 80% sequence identity to the amino acid sequence set forth as any one of SEQ ID NOs: 1-7, or fragments thereof. Recombinant vectors including a nucleic acid encoding the protein (such as a nucleic acid encoding a protein with at least 80% sequence identity to any one of SEQ ID NOs: 1-7 or fragments thereof) operably linked to a heterologous promoter are also disclosed. Also disclosed are methods of identifying compounds that increase β cell number and/or proliferation by determining the effect of test compounds on β cell number or proliferation in zebrafish pancreas.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the § 371 U.S. National Stage of International Application No.PCT/US2015/051744, filed Sep. 23, 2015, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Application No. 62/054,685, filed Sep. 24, 2014, and U.S.Provisional Application No. 62/167,061, filed May 27, 2015, both ofwhich are incorporated herein by reference in their entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant numbers P50GM098911-03A1 and T32 GM007413-37 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

FIELD

This disclosure relates to proteins from gut microbiota, particularlyproteins that increase β-cells in the pancreas, methods of their use,and methods for identifying such proteins.

BACKGROUND

Diabetes is an increasingly common disease, with about 8-9% of the U.S.population diagnosed with diabetes and over 35% of the U.S. populationhaving prediabetes. Individuals with diabetes are at increased risk forheart disease, stroke, blindness, kidney failure, and peripheralneuropathy (and potential loss of lower limbs). The development of bothType I and Type II diabetes is likely to involve a combination ofgenetic and environmental factors. Type I diabetes results fromdestruction of insulin-producing β cells in the pancreas. Treatmentincludes administration of insulin by injection or pump. Type IIdiabetes includes insulin resistance, which gradually leads to adecrease or loss of insulin production by β cells in the pancreas.

SUMMARY

Despite the development of treatments for diabetes, there remains a needfor additional diabetes therapies, particularly for Type I diabetes. Theinventors have surprisingly identified a protein from the gut bacteriumAeromonas that increases the number of pancreatic β cells (for example,increases proliferation, differentiation, and/or survival of pancreaticβ cells), and which could be used to treat or inhibit diabetes in asubject.

Disclosed herein are polypeptides that increase pancreatic β cellnumber. In some embodiments, the polypeptides have at least 80% sequenceidentity to any one of SEQ ID NOs: 1-7 disclosed herein or a fragmentthereof (such as a SYLF domain, for example, a fragment corresponding toamino acids 114-258 of any of SEQ ID NOs: 1-3). Also disclosed arepolynucleotides that encode the polypeptides, including nucleic acidswith at least 80% sequence identity to any one of SEQ ID NOs: 8-11 or afragment thereof (for example, a nucleic acid encoding a SYLF domain,such as a nucleic acid corresponding to nucleotides 340-774 of any oneof SEQ ID NOs: 8-10).

Recombinant vectors including a nucleic acid encoding the hereinidentified proteins that increases pancreatic β cell number (such as anucleic acid encoding a protein with at least 80% sequence identity toany one of SEQ ID NOs: 1-7 or a fragment thereof) operably linked to aheterologous promoter are also disclosed. In some examples, the nucleicacid encoding the protein is set forth in SEQ ID NOs: 8-11. Cellsincluding the recombinant vector (for example, cells transformed withthe vector) are also disclosed.

Disclosed herein are methods of treating or inhibiting diabetes (such astype I diabetes) in a subject by administering to a subject a proteindisclosed herein, a nucleic acid encoding the protein, a cell expressingthe protein, or a composition including the protein. In someembodiments, the protein has at least 80% sequence identity to the aminoacid sequence set forth as any one of SEQ ID NOs: 1-7 or fragmentsthereof. Also disclosed are methods of increasing β cell number orproliferation, by contacting pancreatic cells with a protein disclosedherein, a nucleic acid encoding the protein, a cell expressing theprotein, or a composition including the protein.

Also disclosed herein are methods of identifying compounds that increaseβ cell number by determining the effect of test compounds on β cellnumber or proliferation in zebrafish pancreas. In some embodiments,germ-free zebrafish are contacted with one or more test compounds (suchas a defined bacterial strain, an extract from a defined bacterialstrain, or one or more compounds) and the number of β cells in thepancreas of the zebrafish are measured and compared to a control. In onespecific example, the zebrafish is a transgenic zebrafish expressinggreen fluorescent protein (GFP) or another marker under the control ofthe insulin promoter. This system permits measurement of β cell numberin the pancreas of living organisms.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E are a series of panels showing that bacteria are required fornormal expansion of the β cell mass. FIG. 1A is a graph showing thetotal number of beta cells per larvae in GF (white box plots) and CV(grey box plots) zebrafish at 3, 4, 5 and 6 days post-fertilization(dpf). Single factor ANOVA indicates that gnotobiology of the fish wassignificant in determining the number of β cells present (F₇=9.01,p=1.45e⁻⁸). Labels A, AB and B indicate results of post hoc meanstesting (Tukey). The difference between GF and CV cell counts becamesignificant at 6 dpf (t=−5.91, p<0.001). This significance is consistentacross FIGS. 1B-E. Z-projections of confocal scans through the primaryislet are shown for CV (FIG. 1B) and GF (FIG. 1C) Tg(−1.0insulin:eGFP)larvae age 6 dpf. Cells were stained for insulin (β cells) and nuclei.Scale bar=10 μm. FIG. 1D is a graph showing quantification of β cells inGF larvae treated at 4 dpf with either non-sterile tank water (XGF) ormono-associated with the indicated bacterial strain isolated from thezebrafish gut. Bacterial mono-associations are labeled by genus.Aeromonas sp. are also labeled with a strain identifier (HM21, ZF1 &ZF2). All fish in this, and subsequent figures, were quantified at 6dpf. Data represented in shaded box plots, here and in all subsequentfigures, were found to be significantly greater (p<0.05) than GFcontrols via Tukey analysis. FIG. 1E is a graph showing average amountof glucose (pmol) per larvae aged 6 dpf*t₁₇=−3.65, p<0.01.

In all relevant panels and remaining figures, box plot whiskersrepresent the minimum and maximum values of the data set. Vibrio (*)colonized the zebrafish gut, but did not induce β cell expansion

FIGS. 2A and 2B are graphs showing colony forming units (CFU) inzebrafish gut. FIG. 2A is a graph showing that bacterial isolates of thezebrafish gut are capable of forming mono-associations with larvae. Thegraph shows quantification of the CFU per gut in each strain that wasassayed in FIG. 1D. Shaded box plots denote strains which weresufficient to rescue GF β cell numbers (as shown in FIG. 1D), dashedline denotes limit of detection. FIG. 2B is a graph showing CFU detectedfrom zebrafish with di-association of A. veronii WT and A. veronii ΔbefAstrains. When inoculated in a 1:1 ratio, the WT strain colonized the gutat slightly higher (though not statistically significant) levels thatthe ΔbefA strain. Vibrio (*) colonized the zebrafish gut, but did notinduce β cell expansion

FIGS. 3A-3E are a series of panels showing the effect of a secretedprotein on expansion of the β cell mass. FIG. 3A is a graph showingtotal β cell numbers in GF fish treated at 4 dpf with different samplesof cell free supernatant (CFS). WT represents wild type A. veronii. +PKindicates proteinase K was added to the CFS sample prior to treatment.ΔT2SS represents the A. veronii ^(ΔT2SS) mutant. FIG. 3B is a graphshowing total β cells in GF fish treated with various mono-associations(MA) or samples of CFS. 10165 represents purified protein from theM001_10165 locus (also referred to as BefA). ΔbefA represents the A.veronii ^(ΔbefA) mutant strain. FIGS. 3C-3E are a series of digitalimages of Z-projections of confocal scans through the primary islet ofGF (FIG. 3C), CV (FIG. 3D), and 10165 protein treated (FIG. 3E)Tg(−1.0insulin:eGFP) larvae age 6 dpf stained for insulin and nuclei.Scale bar=10 μm.

FIGS. 4A and 4B are panels showing fractionation of A. veronii CFS. FIG.4A is a graph showing total β cell numbers in GF fish treated at 4 dpfwith separate ammonium sulfate fractions prepared from the A. veronii^(ΔT2SS) CFS. FIG. 4B is a digital image of an SDS-page gel showingsubsequent steps in the purification of BefA (predicted size of 29 kDa,arrow) from E. coli cell lysate, lane 1: ladder, lane 2: cell lysateafter IPTG induction, lane 3: supernatant from cell lysate after theaddition of nickel beads, lane 4: 20 mM imidazole wash step, lanes 5-8:elutions of BefA from nickel beads.

FIGS. 5A-5I are a series of panels showing the effect of BefA on β cellproliferation. FIGS. 5A-5F are images of representative 2D slices fromconfocal scans through the primary islets of CV (FIGS. 5A-5B), GF (FIGS.5C-5D), and BefA-treated (FIGS. 5E-5F) Tg(−1.0insulin:eGFP) larvae age 6dpf showing relative levels of proliferating cells marked with EdU,insulin, and nuclei. Scale bar=10 μm. The perimeter of insulinexpression is marked with a dotted white line (FIGS. 5B, 5D, and 5F).FIG. 5G is a graph showing quantification of the percentage of EdUlabeled β cells per fish. FIG. 5H is a graph showing quantification ofthe percentage of EdU labeled cells from murine β-TC-6 culturesfollowing treatment with BefA or a control. ***t_(43.99)=4.28, p<0.0001.FIG. 5I is representative merged image of β-TC-6 cells stained withnuclei, insulin, and EdU.

FIGS. 6A-6C are a series of panels showing β cell proliferation inzebrafish and mammalian cells. FIG. 6A is a graph showing quantificationof EdU⁺ pancreatic exocrine cells in CV, GF or BefA treated GF fish.FIG. 6B is a graph showing quantification of EdU⁺ intestinal epithelialcells in CV, GF or BefA treated GF fish. FIG. 6C is a representativeimage of IEC-6 cells showing nuclei and EdU.

FIGS. 7A and 7B are phylogenetic trees of homologs of BefA acrossmicrobial species. FIG. 7A shows close homologs of BefA across microbialspecies. Each species is represented by its closest BefA homolog, with aminimum allowed amino acid sequence identity of 50% (relative to thequery sequence). Notably, the Enterococcus gallinarum homolog clustersamong homologs from the Aeromonas genus, which is evidence of a possiblelateral gene transfer event. FIG. 7B shows BefA phylogeny including moredistant homologs (sequence identity >20%) and grouped by genus. Theportion of the tree represented in FIG. 7A is contained in the lightgray box. Enterococcus, Enterobacter, Klebsiella, and Escherichia aregenera that were associated with humans in metagenomes produced duringthe Human Microbiome Project. In both FIGS. 7A and 7B, the numbersindicate branch support (values closer to 1 are better supported);branches with support values <0.5 have been collapsed. Scale barsindicate amino acid substitutions per amino acid site.

SEQUENCE LISTING

Any nucleic acid and amino acid sequences listed herein or in theaccompanying sequence listing are shown using standard letterabbreviations for nucleotide bases and amino acids, as defined in 37C.F.R. § 1.822. In at least some cases, only one strand of each nucleicacid sequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand.

The Sequence Listing is submitted as an ASCII text file in the form ofthe file named Sequence_Listing.txt, which was created on Mar. 16, 2017,and is 19,892 bytes, which is incorporated by reference herein.

SEQ ID NOs: 1-3 are exemplary amino acid sequences of an Aeromonasprotein that increases pancreatic β cell number.

SEQ ID NO: 4 is an exemplary amino acid sequence of a putativeShewanella protein that increases β cell number.

SEQ ID NO: 5 is an exemplary amino acid sequence of a putativeKlebsiella protein that increases β cell number.

SEQ ID NO: 6 is an exemplary amino acid sequence of a putativeEnterococcus gallinarum protein that increases β cell number.

SEQ ID NO: 7 is an exemplary amino acid sequence of a putativePhotobacterium protein that increases β cell number.

SEQ ID NOs: 8-10 is are nucleic acid sequence encoding the amino acidsequence of SEQ ID NOs: 1-3, respectively.

SEQ ID NO: 11 is a nucleic acid sequence encoding the amino acidsequence of SEQ ID NO: 4.

SEQ ID NOs: 12 and 13 are primers for the amplification of the Aeromonasnucleic acid of SEQ ID NO: 8.

DETAILED DESCRIPTION

Type I diabetes is a prevalent and costly disease characterized by theloss of insulin secreting β cells of the endocrine pancreas (Van Belleet al., Physiol. Rev. 91:79-118, 2011). Successful treatments for thedisease, beyond exogenous insulin injections, remain elusive despite thefact that the number of diagnosed cases has been increasing steadilyover the past several decades (Patterson et al., Lancet 373:2027-2033,2009). Modern lifestyle changes, such as antibiotic use and anever-increasing emphasis on cleanliness, have been theorized to reduceenvironmental exposure to unique bacterial symbionts that could beessential to health and development (Blaser and Falkow, Nat. Rev. Micro.7:887-894, 2009). Interestingly, the gastrointestinal-associatedmicrobiota is emerging as a potential major environmental factor in theonset of type I diabetes, though the mechanisms through which bacteriainfluence disease etiology are unknown (Fung et al., Curr.Allergy/Asthma Rep. 12:511-519, 2012).

The microbiota has been shown to play a role in many different aspectsof animal development (Fraune and Bosch, Bioessays 32:571-580, 2010).The pancreas and the intestine are intricately connected, both duringdevelopment and throughout adulthood (Field et al., Dev. Biol.261:197-208, 2003). Furthermore, communication between these two organsis essential in order to regulate metabolic homeostasis. Therefore, itis believed that signals from the intestinal microbiota influence β celldevelopment and/or function. Using the larval zebrafish as a model forpancreas development, and gnotobiotic methods, the inventors show hereinthat the loss of host-associated bacteria results in a decrease in thetotal number of β cells in the developing pancreas. Furthermore,exposure to individual bacterial species results in the restoration of βcell numbers to conventionally reared control levels, and that asecreted bacterial protein is responsible for this effect. The proteinis believed to increase the number of β cells in the pancreas bystimulating β cell proliferation, increasing β cell production ordifferentiation, increasing β cell survival, or a combination thereof.

As described herein, it has surprisingly been found that A. veronii andother bacterial strains in possession of the befA locus exert theirinfluence on the pancreas from their location within the gut, since asecreted product from the gut-associated microbiota has herein beenshown to affect critical developmental processes in a separate organ. Itis important to note that the pancreas and the gut, despite beingphysically separated, rely on vital connections to coordinate thephysiological functions of digestion and metabolic homeostasis. If thesebacterial signals require direct interaction with β cells, they couldreach the pancreas by utilizing these inter-organ connections. Forinstance, the pancreas secretes digestive enzymes into the gut lumenthrough the extra pancreatic duct (Field et al., Dev. Biol. 261:197-208,2003). This duct offers a direct pathway between the pancreas and themicrobiota within the gut lumen. Furthermore, bacteria and bacterialproducts can enter the portal vein, which supplies both the liver andthe pancreas with blood after it leaves the intestine (Minemura et al.,World J. Gastroenterol. 21:1691-172, 2015). Alternatively, the bacterialsignal could act on β cells in an indirect manner, by binding anintermediate host cell type, such as enteroendocrine cells of theintestine (Cani et al., Curr. Opin. Pharmacol. 13:935-940, 2013).

Several studies have shown that fecal bacterial communities of diabeticchildren are less diverse than those of healthy (non-diabetic) children(Giongo et al., ISME J. 5:82-91, 2011; Kostic et al., Cell Host Microbe17:260-273, 2015; Murri et al., BMC Med. 11:46, 2013; Mejia-Leon et al.,Sci. Rep. 4:3814, 2014). Even more striking, there is evidence that thisshift in the bacterial community occurs prior to disease onset (Kosticet al., Cell Host Microbe 17:260-273, 2015). It is shown herein that animportant host developmental process involves the presence of a proteinproduced only in a subset of bacterial species. It is possible that thefailure to maintain microbial diversity, as has been shown in patientswith type I diabetes, could result in the loss of important bacterialsignals required for robust β cell development early in life.

I. Abbreviations

BefA β cell expansion factor A

CFS cell-free supernatant

CFU colony-forming units

CV conventionally reared

dpf days post-fertilization

EdU 5-ethynyl-2′-deoxyuridine

EM embryo medium

EPD extra-pancreatic duct

GF germ-free reared

GFP green fluorescent protein

T2SS type 2 secretion system

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Unless otherwise explained, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this disclosurebelongs. The singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Although methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present disclosure, suitablemethods and materials are described below.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Beta (β) cells: A type of cell in the pancreas that secretes insulin.These cells also secrete C-peptide and amylin in addition to insulin.The β cells are present in the islets of Langerhans in the pancreas andmake up about 65-80% of the islets.

Diabetes mellitus: A disease caused by a relative or absolute lack ofinsulin leading to uncontrolled carbohydrate metabolism, commonlysimplified to “diabetes.” As used herein, “diabetes” refers to diabetesmellitus, unless otherwise indicated. A “diabetic condition” includespre-diabetes and diabetes. Type 1 diabetes (sometimes referred to as“insulin-dependent diabetes” or “juvenile-onset diabetes”) is anauto-immune disease characterized by destruction of the pancreatic βcells that leads to a total or near total lack of insulin. In type 2diabetes (sometimes referred to as “non-insulin-dependent diabetes” or“adult-onset diabetes”), the body does not respond to insulin, though itis present.

Symptoms of diabetes include: excessive thirst (polydipsia); frequenturination (polyuria); extreme hunger or constant eating (polyphagia);unexplained weight loss; presence of glucose in the urine (glycosuria);tiredness or fatigue; changes in vision; numbness or tingling in theextremities (hands, feet); slow-healing wounds or sores; and abnormallyhigh frequency of infection. Diabetes may be clinically diagnosed by afasting plasma glucose concentration of greater than or equal to 7.0mmol/L (126 mg/dL), or a plasma glucose concentration of greater than orequal to 11.1 mmol/L (200 mg/dL) at about two hours after an oralglucose tolerance test with a 75 g load. A more detailed description ofdiabetes may be found in Cecil Textbook of Medicine, Goldman, et al.,eds. (Elsevier, 2003, 22^(nd) ed.).

Effective amount: An amount of an agent or composition that alone, ortogether with a pharmaceutically acceptable carrier and/or one or moreadditional agents, induces the desired response. In some embodiments, aneffective amount is an amount that increases pancreatic β cell number ofproliferation or an amount that delays, reduces, or ameliorates or onemore symptoms of diabetes in a subject. Effective amounts of an agentcan be determined in many different ways, such as assaying for cellnumber (such asp cell number or proliferation, for example at least a10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more increase in(cell number or proliferation), delay (or even prevention) of onset of acondition associated with β cells (such as diabetes), or a reduction oramelioration of one or more symptoms of a subject with diabetes (such asat least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or morereduction in one or more symptoms of diabetes). Effective amounts alsocan be determined through various in vitro, in vivo or in situ assays,including, but not limited to those described herein.

Germ-free: An animal born and reared in aseptic conditions havingsubstantially no microorganisms living on or in it (for example,substantially no bacteria in the gut of the animal).

Gnotobiotic: An animal in which only known strains of microorganisms arepresent. For example, a germ-free animal exposed to (e.g., intentionallyinoculated with) one or more known bacterial strains is gnotobiotic.Germ-free animals are also gnotobiotic, as their microbial status isknown. In contrast, conventionally reared animals (born and raisedwithout absolute control of microorganism exposure) have a microbiota ofmany, and in most cases hundreds or thousands of organisms, whichpopulation will vary from animal to animal.

Heterologous: Originating from a different genetic sources or species.For example, a nucleic acid that is heterologous to a cell originatesfrom an organism or species other than the cell in which it isexpressed. In one specific, non-limiting example, a heterologous nucleicacid includes an Aeromonas nucleic acid that is present or expressed ina different bacterial cell (such as an E. coli cell) or in an algal,plant, or mammalian cell. Methods for introducing a heterologous nucleicacid into bacterial, algal, plant, and mammalian cells are well known inthe art, for example transformation with a nucleic acid, includingelectroporation, lipofection, and particle gun acceleration.

In another example of use of the term heterologous, a nucleic acidoperably linked to a heterologous promoter is from an organism orspecies other than that of the promoter. For example, an Aeromonasnucleic acid may be linked to a heterologous bacterial, viral, ormammalian promoter. In other examples of the use of the termheterologous, a nucleic acid encoding a polypeptide (such as a proteinincreasing β cell number disclosed herein) or portion thereof isoperably linked to a heterologous nucleic acid encoding a secondpolypeptide or portion thereof, for example to form a non-naturallyoccurring fusion protein.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein, or cell) has been substantially separated or purifiedaway from other biological components in the cell of the organism, orthe organism itself, in which the component naturally occurs, such asother chromosomal and extra-chromosomal DNA and RNA, proteins and/orcells. Nucleic acid molecules and proteins that have been “isolated”include nucleic acid molecules and proteins purified by standardpurification methods or prepared by recombinant expression in a hostcell, as well as chemically synthesized nucleic acid molecules andproteins.

Operably linked: A first nucleic acid is operably linked with a secondnucleic acid when the first nucleic acid is placed in a functionalrelationship with the second nucleic acid. For instance, a promoter isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

Pharmaceutically acceptable carrier: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington: TheScience and Practice of Pharmacy, The University of the Sciences inPhiladelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia,Pa., 21^(st) Edition (2005), describes compositions and formulationssuitable for pharmaceutical delivery of one or more therapeutic agents,such as those disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol, or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, pH buffering agents, or the like, for example sodiumacetate or sorbitan monolaurate.

Proliferation: An increase in cell number, for example by cell division.

Recombinant: A nucleic acid or protein that is not naturally occurringor has a sequence that is made by an artificial combination of twootherwise separated segments of nucleotides or amino acids. Thisartificial combination is often accomplished by chemical synthesis or,more commonly, by the artificial manipulation of isolated segments ofnucleic acids, e.g., by genetic engineering techniques such as thosedescribed in Sambrook et al. Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Laboratory Press, NY, 2001. The termrecombinant includes nucleic acids or proteins that have been alteredsolely by addition, substitution, or deletion of a portion of thenucleic acid sequence or amino acid sequence, respectively.

Sample (or biological sample): A specimen containing genomic DNA, RNA(including mRNA), protein, or combinations thereof, obtained from asubject. Examples include, but are not limited to, peripheral blood (orfractions thereof), fine needle aspirate, urine, saliva, feces, tissuebiopsy, surgical specimen, and autopsy material.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations. The NCBI BasicLocal Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol.215:403-10, 1990) is available from several sources, including theNational Center for Biotechnology (NCBI, National Library of Medicine,Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, foruse in connection with the sequence analysis programs blastp, blastn,blastx, tblastn and tblastx. Additional information can be found at theNCBI web site.

One of skill in the art will appreciate that the particular sequenceidentity ranges provided herein are for guidance only; it is possiblethat strongly significant homologs or orthologs could be obtained thatfall outside the ranges provided.

Subject: Living multi-cellular vertebrate organism, a category thatincludes vertebrates, including human and non-human mammals. In someexamples, a subject includes laboratory animals, including mice orzebrafish.

SYLF domain: Also referred to as DUF500. A highly conservedlipid-binding (for example, phosphoinositide-binding) module present inproteins from bacteria to mammals. See, e.g., NCBI Conserved DomainsDatabase Accession No. cd11524, incorporated herein by reference aspresent in the database on May 27, 2015.

Transduced and Transformed: A virus or vector “transduces” a cell whenit transfers nucleic acid into the cell. A cell is “transformed” by anucleic acid transduced into the cell when the DNA becomes replicated bythe cell, either by incorporation of the nucleic acid into the cellulargenome, or by episomal replication. As used herein, the termtransformation encompasses all techniques by which a nucleic acidmolecule might be introduced into such a cell, including transfectionwith viral vectors, transformation with plasmid vectors, andintroduction of naked DNA by electroporation, lipofection, and particlegun acceleration.

Treating or Inhibiting: “Inhibiting” refers to inhibiting or reducingthe full development of a condition or disorder (such as diabetes) orone or more symptoms thereof. Inhibition of a condition or disorder canspan the spectrum from partial inhibition (reduction) to substantiallycomplete inhibition (prevention) of the condition or disorder or one ormore symptoms thereof. In some examples, the term “inhibiting” refers toreducing or delaying the onset or progression of diabetes. In contrast,“treatment” refers to a therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition (such as diabetes)after it has begun to develop.

Vector: A nucleic acid molecule that can be introduced into a host cell,thereby producing a transformed or transduced host cell. Recombinant DNAvectors are vectors including recombinant DNA. A vector can includenucleic acid sequences that permit it to replicate in a host cell, suchas an origin of replication. A vector can also include one or moreselectable marker genes, a cloning site for introduction of heterologousnucleic acids, a promoter (for example for expression of an operablylinked nucleic acid), and/or other genetic elements known in the art.Vectors include plasmid vectors, including plasmids for expression ingram negative and gram positive bacterial cell. Exemplary vectorsinclude those for use in E. coli. Vectors also include viral vectors,such as, but not limited to, retrovirus, orthopox, avipox, fowlpox,capripox, suipox, adenovirus, herpes virus, alpha virus, baculovirus,Sindbis virus, vaccinia virus, and poliovirus vectors. Vectors alsoinclude vectors for expression in yeast cells or mammalian cells.

In some examples, a heterologous nucleic acid (such as a nucleic acidencoding an Aeromonas protein) is introduced into a vector to produce arecombinant vector, thereby allowing the nucleic acid to be renewablyproduced and or a protein encoded by the nucleic acid to be expressed.

III. Proteins Increasing β Cell Number

Disclosed herein are proteins from members of the intestinal microbiota,including Aeromonas, Shewanella, Klebsiella, Enterococcus,Photobacterium, and/or other bacteria that increase the number of βcells in the pancreas of a subject to which the protein is provided. Inparticular examples, the proteins increase β cell proliferation. Inother examples, the proteins increase β cell number by mechanisms otherthan, or in addition to, increasing β cell proliferation.

In some embodiments the protein is a polypeptide which includes,consists essentially of, or consists of the amino acid sequence setforth as any one of SEQ ID NOs: 1-3. In additional embodiments, apolypeptide increasing β cell number disclosed herein has at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%sequence identity to the amino acid sequence set forth in SEQ ID NOs:1-3, for example from Aeromonas (such as A. veronii) or Shewanella (suchas S. oneidensis). In some examples herein, the protein is referred toas BefA (and the corresponding nucleic acid is referred to as befA).Exemplary sequences can be obtained using computer programs that arereadily available on the internet and the amino acid sequences set forthherein. In some examples, the polypeptide retains a function of thedisclosed protein, such as increasing the number of β cells and/orreducing or inhibiting one or more signs or symptoms of diabetes (suchas type I diabetes) in a subject.

In additional embodiments, a protein increasing β cell numbers (such asSEQ ID NOs: 1-3) includes a portion or fragment of the protein (forexample, a portion of an Aeromonas protein disclosed herein). In someexamples, the protein or portion or fragment thereof includes at least20 contiguous amino acids of a disclosed protein, for example, at least30, at least 50, at least 75, at least 100, at least 150, at least 200,at least 250, or more amino acids of a protein increasing β cell number,such as SEQ ID NOs: 1-3. In other examples, the protein or portionthereof includes at least the first half, the second half, at least onethird, at least one quarter, at least one fifth of the protein, or anycombination thereof (such as at least two thirds, at least two quarters,at least three fifths, and so on). In other examples, a portion orfragment of a protein increasing β cell number includes one or moredomains of the protein. In some examples, a domain may include a SYLFdomain, such as amino acids 114-258 of any one of SEQ ID NOs: 1-3. Insome examples, the protein or a fragment thereof includes, consistsessentially of, or consists of a SYLF domain. In other examples, theprotein or fragment thereof is a processed or mature protein, forexample, a protein lacking the putative secretion signal sequencecorresponding to amino acids 1-21 of SEQ ID NO: 1). One of ordinaryskill in the art will recognize that the boundaries of a domain (such asa SYLF domain or a secretion signal sequence) is not exact, and in someexamples may include additional or fewer amino acids (for example, about20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1more or less amino acids from either end of the domain). In someexamples, the fragment of the polypeptide retains a function of thedisclosed proteins, such as increasing the number of β cells and/orreducing or inhibiting one or more signs or symptoms of diabetes (suchas type I diabetes) in a subject. One of ordinary skill in the art canidentify the corresponding domains from other proteins that increase βcell numbers, for example a protein from another bacterium or otherorganism.

Exemplary Aeromonas proteins that increase β cell number include theamino acid sequences of GenBank Accession Nos. ERF65753 (SEQ ID NO: 1),YP_004391747, WP_019445797, WP_005334756, WP_005362069, WP_005347598,WP_026456068, WP_019839625, and WP_005298756; all of which areincorporated herein by reference as present in GenBank on Sep. 22, 2014.

Additional proteins increasing β cell number include proteins with atleast 50% (such as at least 55%, 60%, 65%, 70%, 75%, or more identity)sequence identity to SEQ ID NOs: 1-3 or at least 20% (such as at least30%, 40%, 50%, 60%, 70%, 80%, or more identity) to a fragment of SEQ IDNOs: 1-3, such as the SYLF domain (corresponding to amino acids 114-258of SEQ ID NOs: 1-3). Exemplary proteins include a protein including,consisting essentially of, or consisting of SEQ ID NOs: 4-7. Otherexamples include Photobacterium proteins having the amino acid sequencesof GenBank Accession Nos. WP_007468440 (SEQ ID NO: 7) and GAL02513 andVibrio proteins having the amino acid sequences of GenBank AccessionNos. WP_025555898 and KED81752, all of which are incorporated herein byreference as present in GenBank on Sep. 22, 2014. One of ordinary skillin the art can identify additional proteins increasing β cell number,for example from other microbiota, for example utilizing the methodsdescribed below.

Minor modifications of an Aeromonas (or another microbial) proteinincreasing β cell number primary amino acid sequence disclosed herein(such as SEQ ID NOs: 1-7) may result in polypeptides which havesubstantially equivalent activity as compared to the unmodifiedcounterpart polypeptide described herein. Such modifications may bedeliberate, for example as by site-directed mutagenesis (for example,introducing non-naturally occurring changes to the amino acid sequenceor structure), or may be spontaneous. All of the polypeptides producedby these modifications are included herein. Thus, a specific,non-limiting example of an Aeromonas protein increasing β cell numbersis a conservative variant of the protein (such as a single conservativeamino acid substitution, for example, one or more conservative aminoacid substitutions, for example 1-10 conservative substitutions, 2-5conservative substitutions, 4-9 conservative substitutions, such as 1,2, 5 or 10 conservative substitutions). In other examples, the Aeromonasprotein increasing β cell number may include one or morenon-conservative substitutions (for example 1-10 non-conservativesubstitutions, 2-5 non-conservative substitutions, 4-9 non-conservativesubstitutions, such as 1, 2, 5 or 10 non-conservative substitutions), solong as the protein retains an activity of increasing β cell number inthe pancreas.

In additional embodiments, the protein includes a tag (such as anN-terminal or C-terminal tag), for example for use in proteinpurification. One of skill in the art can select appropriate tags, suchas a His-tag, a GST tag, or an antibody recognition sequence (such as aMyc-tag or HA-tag). In some examples, the tag is removed prior to us(for example, prior to administration to a subject). The protein canalso be produced as a fusion protein, either to facilitate expressionand/or purification or to facilitate delivery to a subject. For example,fusion proteins including a therapeutic molecule (such as the disclosedproteins) and transferrin has been shown to be useful for oral deliveryroutes. In other examples, the disclosed proteins may include adetectable label, such as a radioisotope, fluorophore, or hapten.

In some embodiments, the Aeromonas protein increasing β cell number isencoded by a nucleic acid sequence which includes, consists essentiallyof, or consists of the nucleic acid sequence set forth as SEQ ID NOs:8-10. In additional embodiments, a nucleic acid encoding an Aeromonasprotein increasing β cell number disclosed herein has at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to the nucleic acid sequence set forth in SEQ ID NOs: 8-10 or afragment thereof. In some examples, the nucleic acid has a nucleic acidsequence with at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity to a nucleic acid encodinga portion of a protein increasing β cell number, such as encoding theSYLF domain, for example corresponding to amino acids 114-258 of SEQ IDNOs: 1-3 (such as corresponding to nucleotides 340-774 of SEQ ID NOs:8-10) or encoding a mature (processed) form of the protein (such ascorresponding to nucleotides 64-786 of SEQ ID NO: 8). Exemplarysequences can be obtained using computer programs that are readilyavailable on the internet and the nucleic acid and amino acid sequencesset forth herein. In some examples, the nucleic acid encodes apolypeptide that retains a function of the protein, such as increasingthe number of β cells and/or reducing or inhibiting one or more signs orsymptoms of diabetes in a subject.

Exemplary Aeromonas nucleic acids encoding the disclosed proteinsinclude SEQ ID NOs: 8-10 and the nucleic acid sequences of GenBankAccession Nos. NZ_ATFB01000020 (nucleotides 294943 . . . 295728) (SEQ IDNO: 8), CP002607 (nucleotides 1210185 . . . 1210970), NZ_ALOT01000039(complement7714 . . . 8499), NZ_JMGO01000024 (nucleotides 4145 . . .4924), and NZ_AOTK01000020 (complement 37734 . . . 38510); all of whichare incorporated herein by reference in their entirety as present inGenBank on Sep. 22, 2014.

Additional nucleic acids encoding proteins increasing β cell numberinclude nucleic acids with at least 50% (such as at least 55%, 60%, 65%,70%, 75%, or more identity) sequence identity to SEQ ID NOs: 8-10 or atleast 20% (such as at least 30%, 40%, 50%, 60%, 70%, 80%, or moreidentity) to a fragment of SEQ ID NOs: 8-10, such as the SYLF domain(e.g., corresponding to nucleotides 340-774 of SEQ ID NOs: 8-10) or amature (processed) form of the protein (such as corresponding tonucleotides 64-786 of SEQ ID NO: 8). An additional exemplary additionalnucleic acid includes, consists essentially of, or consists of SEQ IDNO: 11. In some examples, the nucleic acid sequences include those ofGenBank Accession Nos. NZ_AMZO01000030 (nucleotides 1616 . . . 2386) andBBMN01000001 (nucleotides 356894 . . . 357667) (Photobacterium) orNZ_AWMU01000020 (complement of nucleotides 20075 . . . 20854) andINTF01000013 (nucleotides 123187 . . . 123966) (Vibrio), NZ_KI535451(complement of nucleotides 317490 . . . 318077, Klebsiella), andACOV01006014 (Enterococcus gallinarum), all of which are incorporatedherein by reference in their entirety as present in GenBank on May 27,2015. One of ordinary skill in the art can identify additional nucleicacids increasing β cell number related to the proteins disclosed herein,for example from other microbiota.

Minor modifications of nucleic acids encoding a protein increasing βcell number primary amino acid sequence (such as SEQ ID NOs: 8-11) arealso contemplated herein. Such modifications to the nucleic acid mayresult in polypeptides that have substantially equivalent activity ascompared to the unmodified counterpart polypeptide described herein.Such modifications may be deliberate, for example as by site-directedmutagenesis, or may be spontaneous. All of the nucleic acids produced bythese modifications are included herein. Thus, a specific, non-limitingexample of modified nucleic acid encoding protein increasing β cellnumber is a nucleic acid encoding conservative variant of the protein(such as a single conservative amino acid substitution, for example, oneor more conservative amino acid substitutions, for example 1-10conservative substitutions, 2-5 conservative substitutions, 4-9conservative substitutions, such as 1, 2, 5 or 10 conservativesubstitutions). In other examples, the nucleic acid may encode a proteinincluding one or more non-conservative substitutions (for example 1-10non-conservative substitutions, 2-5 non-conservative substitutions, 4-9non-conservative substitutions, such as 1, 2, 5 or 10 non-conservativesubstitutions), so long as the encoded protein retains activity thatincreases β cell number or β cell proliferation.

In some examples, the nucleic acid encoding the protein increasing βcell number is codon-optimized for the cell in which it is to beexpressed. Codon usage bias, the use of synonymous codons at unequalfrequencies, is ubiquitous among genetic systems (Ikemura, J. Mol. Biol.146:1-21, 1981; Ikemura, J. Mol. Biol. 158:573-97, 1982). The strengthand direction of codon usage bias is related to genomic G+C content andthe relative abundance of different iso accepting tRNAs (Akashi, Curr.Opin. Genet. Dev. 11:660-6, 2001; Duret, Curr. Opin. Genet. Dev.12:640-9, 2002; Osawa et al., Microbiol. Rev. 56:229-64, 1992). Codonusage can affect the efficiency of gene expression. For example, inEscherichia coli (Ikemura, J. Mol. Biol. 146:1-21, 1981; Xia Genetics149:37-44, 1998) the most highly expressed genes use codons matched tothe most abundant tRNAs (Akashi and Eyre-Walker, Curr. Opin. Genet. Dev.8:688-93, 1998).

Codon-optimization refers to replacement of a codon in a nucleic acidsequence with a synonymous codon (one that codes for the same aminoacid) more frequently used (preferred) in the organism. Each organismhas a particular codon usage bias for each amino acid, which can bedetermined from publicly available codon usage tables (for example seeNakamura et al., Nucleic Acids Res. 28:292, 2000 and references citedtherein). For example, a codon usage database is available on the WorldWide Web at kazusa.or.jp/codon. One of skill in the art can modify anucleic acid encoding a particular amino acid sequence, such that itencodes the same amino acid sequence, while being optimized forexpression in a particular cell type (such as a bacterial or mammaliancell). However, one of skill in the art will recognize that a nucleicacid does not have to be optimized for expression in a particularorganism in order to be used for gene expression in the selectedorganism.

In additional embodiments, the nucleic acid encoding the proteinincreasing β cell number further includes a nucleic acid sequenceencoding a tag (such as an N-terminal or C-terminal tag), for examplefor use in protein purification. One of skill in the art can selectnucleic acids encoding appropriate tags, such as a His-tag, a GST tag,or an antibody recognition sequence (such as a Myc-tag or HA-tag). Thenucleic acid may also encode a fusion, for example, a nucleic acidencoding a fusion protein including a disclosed protein and transferrin.In other examples, the disclosed nucleic acids may include a detectablelabel, such as a radioisotope, fluorophore, or hapten.

Nucleic acid molecules encoding a protein increasing β cell numberdisclosed herein also include a recombinant DNA which is incorporatedinto a vector, into an autonomously replicating plasmid or virus, orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (such as a cDNA) independent of other sequences. Anucleic acid encoding a protein increasing β cell number (such as anucleic acid encoding a protein increasing β cell number, for exampleany one of SEQ ID NOs: 8-11 or a fragment thereof) is in some examplesoperatively linked to heterologous expression control sequences. Anexpression control sequence operatively linked to a coding sequence isligated such that expression of the coding sequence is achieved underconditions compatible with the expression control sequences. Theexpression control sequences include, but are not limited to,appropriate promoters, enhancers, transcription terminators, a startcodon (e.g., ATG) in front of a protein-encoding nucleic acid, splicingsignal for introns, maintenance of the correct reading frame of thatgene to permit proper translation of mRNA, and stop codons. Theexpression control sequence(s) in some examples are heterologousexpression control sequence(s), for example from an organism or speciesother than the protein-encoding nucleic acid. Thus, the protein-encodingnucleic acid operably linked to a heterologous expression controlsequence (such as a promoter) comprises a nucleic acid that is notnaturally occurring. In other examples, the nucleic acid is operablylinked to a tag sequence (such as 6× His, HA tag, or Myc tag) or anotherprotein-coding sequence, such as glutathione S-transferase or maltosebinding protein.

Vectors for cloning, replication, and/or expression of the disclosednucleic acid molecules include bacterial plasmids, such as bacterialcloning or expression plasmids (some of which can be used for expressionin bacterial and/or mammalian cells). Exemplary bacterial plasmids intowhich the nucleic acids can be cloned include E. coli plasmids, such aspBR322, pUC plasmids (such as pUC18 or pUC19), pBluescript, pACYC184,pCD1, pGEM® plasmids (such as pGEM®-3, pGEM®-4, pGEM-T® plasmids;Promega, Madison, Wis.), TA-cloning vectors, such as pCR® plasmids (forexample, pCR® II, pCR® 2.1, or pCR® 4 plasmids; Life Technologies, GrandIsland, N.Y.) or pcDNA plasmids (for example pcDNA™ 3.1 or pcDNA™ 3.3plasmids; Life Technologies). In some examples, the vector includes aheterologous promoter which allows protein expression in bacteria.Exemplary vectors include pET vectors (for example, pET-21b), pDEST™vectors (Life Technologies), pRSET vectors (Life Technologies), pBADvectors, and pQE vectors (Qiagen). The disclosed nucleic acids can bealso be cloned into B. subtilis plasmids, for example, pTA1060 and pHTplasmids (such as pHT01, pHT43, or pHT315 plasmids). One of skill in theart can select additional vectors suitable for cloning and/or bacterialor mammalian expression of proteins increasing (cell number such asthose disclosed herein.

In other embodiments, vectors are used for expression in yeast such asS. cerevisiae or Kluyveromyces lactis. Several promoters are known to beof use in yeast expression systems such as the constitutive promotersplasma membrane H⁺-ATPase (PMA1), glyceraldehyde-3-phosphatedehydrogenase (GPD), phosphoglycerate kinase-1 (PGK1), alcoholdehydrogenase-1 (ADH1), and pleiotropic drug-resistant pump (PDR5). Inaddition, many inducible promoters are of use, such as GAL1-10 (inducedby galactose), PHO5 (induced by low extracellular inorganic phosphate),and tandem heat shock HSE elements (induced by temperature elevation to37° C.). Promoters that direct variable expression in response to atitratable inducer include the methionine-responsive MET3 and MET25promoters and copper-dependent CUP1 promoters. Any of these promotersmay be cloned into multicopy (2μ) or single copy (CEN) plasmids to givean additional level of control in expression level. The plasmids caninclude nutritional markers (such as URA3, ADE3, HIS1, and others) forselection in yeast and antibiotic resistance (such as AMP) forpropagation in bacteria. Plasmids for expression on K. lactis are known,such as pKLAC1. Thus, in one example, after amplification in bacteria,plasmids can be introduced into the corresponding yeast auxotrophs bymethods similar to bacterial transformation.

Viral vectors including the disclosed polynucleotides (such aspolynucleotides encoding a protein increasing β cell number) can also beprepared. A number of viral vectors have been constructed, includingpolyoma, SV40 (Madzak et al., 1992, J. Gen. Virol., 73:15331536),adenovirus (Berkner, 1992, Curr. Top. Microbiol. Immunol., 158:39-6;Berliner et al., 1988, BioTechniques, 6:616-629; Gorziglia et al., 1992,J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Natl. Acad. Sci.USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155; Wilkinsonet al., 1992, Nucl. Acids Res., 20:2233-2239; Stratford-Perricaudet etal., 1990, Hum. Gene Ther., 1:241-256), vaccinia virus (Mackett et al.,1992, Biotechnology, 24:495-499), adeno-associated virus (Muzyczka,1992, Curr. Top. Microbiol. Immunol., 158:91-123; On et al., 1990, Gene,89:279-282), herpes viruses including HSV and EBV (Margolskee, 1992,Curr. Top. Microbiol. Immunol., 158:67-90; Johnson et al., 1992, J.Virol., 66:2952-2965; Fink et al., 1992, Hum. Gene Ther. 3:11-19;Breakfield et al., 1987, Mol. Neurobiol., 1:337-371; Fresse et al.,1990, Biochem. Pharmacol., 40:2189-2199), Sindbis viruses (Herweijer etal., 1995, Hum. Gene Ther. 6:1161-1167; U.S. Pat. Nos. 5,091,309 and5,2217,879), alphaviruses (S. Schlesinger, 1993, Trends Biotechnol.11:18-22; Frolov et al., 1996, Proc. Natl. Acad. Sci. USA93:11371-11377) and retroviruses of avian (Brandyopadhyay et al., 1984,Mol. Cell Biol., 4:749-754; Petropouplos et al., 1992, J. Virol.,66:3391-3397), murine (Miller, 1992, Curr. Top. Microbiol. Immunol.,158:1-24; Miller et al., 1985, Mol. Cell Biol., 5:431-437; Sorge et al.,1984, Mol. Cell Biol., 4:1730-1737; Mann et al., 1985, J. Virol.,54:401-407), and human origin (Page et al., 1990, J. Virol.,64:5370-5276; Buchschalcher et al., 1992, J. Virol., 66:2731-2739).Baculovirus (Autographa californica multinuclear polyhedrosis virus;AcMNPV) vectors are also known in the art, and may be obtained fromcommercial sources (such as PharMingen, San Diego, Calif.; ProteinSciences Corp., Meriden, Conn.; Stratagene, La Jolla, Calif.).

DNA sequences encoding a protein increasing β cell number can beexpressed in vitro by DNA transfer into a suitable host cell. The cellmay be prokaryotic or eukaryotic. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. Methods of stable transfer, meaning that the foreignDNA is continuously maintained in the host, are known in the art.

Host cells can include microbial, yeast, insect and mammalian hostcells. Methods of expressing DNA sequences having eukaryotic or viralsequences in prokaryotes are well known in the art. Non-limitingexamples of suitable host cells include bacteria, Archaea, insect, fungi(for example, yeast), mycobacterium (such as M. smegmatis), plant, andanimal cells (for example, mammalian cells, such as human). Exemplarycells of use include E. coli, Bacillus subtilis, Saccharomycescerevisiae, Salmonella typhimurium, SF9 cells, C129 cells, 293 cells,Neurospora, and immortalized mammalian myeloid and lymphoid cell lines.Techniques for the propagation of mammalian cells in culture arewell-known (see, Jakoby and Pastan (eds.), 1979, Cell Culture. Meth.Enzymol., volume 58, Academic Press, Inc., Harcourt Brace Jovanovich,N.Y.). Examples of commonly used mammalian host cell lines are VERO andHeLa cells, CHO cells, and WI38, BHK, and COS cell lines, although othercell lines may be used, such as cells designed to provide higherexpression, desirable glycosylation patterns, or other features. Asdiscussed above, techniques for the transformation of yeast cells, suchas polyethylene glycol transformation, protoplast transformation andgene guns are also known in the art (see Gietz and Woods Meth. Enzymol.350: 87-96, 2002).

Transformation of a host cell with recombinant DNA can be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as, but not limited to, E. coli,competent cells which are capable of DNA uptake can be prepared fromcells harvested after exponential growth phase and subsequently treatedby the CaCl₂ method using procedures well known in the art.Alternatively, MgCl₂ or RbCl can be used. Transformation can also beperformed after forming a protoplast of the host cell if desired, or byelectroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors can be used. Eukaryotic cells can also beco-transformed with a polynucleotide encoding a protein increasing βcell number (or a portion or fragment thereof) and a second foreign DNAmolecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982).

IV. Methods of Increasing Pancreatic β Cells

Disclosed herein are methods of increasing pancreatic β cellproliferation or number. In some embodiments, the methods includecontacting pancreatic β cells with an effective amount of a proteinincreasing β cell number (such as a protein with at least 80% sequenceto any one of SEQ ID NOs: 1-7 or a fragment thereof), including, but notlimited to the proteins disclosed herein, cells producing the protein, acell extract, or a preparation (such as a cell-free supernatant) from acell producing the protein, an isolated or purified protein increasing βcell number (including, but not limited to a polypeptide with at least80% sequence identity to any one of SEQ ID NOs: 1-7 or a fragmentthereof), or a nucleic acid encoding a protein increasing β cell number(including, but not limited to, nucleic acids with at least 80% sequenceidentity to any one of SEQ ID NOs: 8-11 or a fragment thereof). In someexamples, contacting pancreatic β cells with the protein or otherpreparation includes administering the preparation to a subject, forexample, a subject with diabetes.

Also disclosed are methods of treating or inhibiting diabetes in asubject. In some embodiments, the methods include administering to asubject an effective amount of a protein increasing β cell number (suchas an Aeromonas protein increasing β cell number, such as a protein withat least 80% sequence to any one of SEQ ID NOs: 1-7 or a fragmentthereof), including, but not limited to the proteins disclosed herein.The protein increasing β cell number may be administered in any form,including administration of cells producing a protein increasing β cellnumber disclosed herein (e.g., A. veronii or other bacteriarecombinantly expressing or overexpressing a protein increasing β cellnumber), a cell extract, or a preparation (such as a cell-freesupernatant) from a cell producing a protein increasing β cell number,an isolated or purified protein increasing β cell number (including, butnot limited to a protein with at least 80% sequence to any one of SEQ IDNOs: 1-7 or a fragment thereof), or a nucleic acid encoding a proteinincreasing β cell number (including, but not limited to, a nucleic acidwith at least 80% sequence to any one of SEQ ID NOs: 8-11 or a fragmentthereof). In particular embodiments, the subject is a subject with typeI diabetes.

The proteins disclosed herein can be chemically synthesized by standardmethods, or can be produced recombinantly. An exemplary process forpolypeptide production is described in Lu et al., FEBS Lett. 429:31-35,1998. They can also be isolated by methods including preparativechromatography and immunological separations. Polypeptides can also beproduced using molecular genetic techniques, such as by inserting anucleic acid encoding a protein increasing β cell number or a portionthereof into an expression vector, introducing the expression vectorinto a host cell (such as E. coli), and isolating the polypeptide (forexample, as discussed in Section III). In some examples, the proteinincludes a tag (such as an N-terminal or C-terminal tag), for examplefor use in protein purification. One of skill in the art can selectappropriate tags, such as a His-tag, a GST tag, or an antibodyrecognition sequence (such as a Myc-tag or HA-tag).

In some embodiments, the protein increasing β cell number (such as aprotein comprising the sequence of any one of SEQ ID NOs: 1-7 or aprotein that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 99% identical to any one of SEQ ID NOs: 1-7 or a fragmentthereof) is administered to a subject to treat or inhibit diabetes. Insome examples, a fragment of the protein includes the processed matureprotein (for example, a protein lacking the putative secretion signalsequence corresponding to amino acids 1-21 of SEQ ID NO: 1) or a domainof the protein (such as a SYLF domain, for example a domaincorresponding to amino acids 114-258 of any one of SEQ ID NOs: 1-3). Inparticular embodiments, the subject has type I diabetes.

The cells, cell extract, protein increasing β cell number, or nucleicacid encoding the protein increasing β cell number can be administeredto a subject in need of treatment using any suitable means known in theart. Methods of administration include, but are not limited to,intradermal, intramuscular, intraperitoneal, parenteral, subcutaneous,rectal, intranasal, inhalation, oral, or by gene gun. Intranasaladministration refers to delivery of the compositions into the nose andnasal passages through one or both of the nares and can include deliveryby a spraying mechanism or droplet mechanism, or through aerosolizationof the therapeutic agent. In particular examples, the protein increasingβ cell number, nucleic acid encoding the protein, or a preparationincluding the protein (such as a cell extract or preparation of cellsexpressing the protein) is administered orally. In other examples, theprotein increasing β cell number, nucleic acid encoding the protein, ora preparation including the protein (such as a cell extract orpreparation of cells expressing the protein) is administeredsubcutaneously or intramuscularly.

Therapeutic agents can be administered in any suitable manner, forexample, with pharmaceutically acceptable carriers. Pharmaceuticallyacceptable carriers are determined in part by the particular compositionbeing administered, as well as by the particular method used toadminister the composition. Accordingly, there is a wide variety ofsuitable formulations of pharmaceutical compositions of the presentdisclosure. Pharmaceutically acceptable carriers (vehicles) useful inthis disclosure are conventional. Remington: The Science and Practice ofPharmacy, The University of the Sciences in Philadelphia, Editor,Lippincott, Williams, & Wilkins, Philadelphia, Pa., 21^(st) Edition(2005), describes compositions and formulations suitable forpharmaceutical delivery of one or more therapeutic agents.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

The amount of protein increasing β cell number, nucleic acid encodingthe protein increasing (3 cell number, or a preparation including theprotein increasing β cell number (such as a cell extract or preparationof cells expressing the protein) to be administered to a subject can beselected by one of ordinary skill in the art, for example from about 1μg to 5 g of the protein increasing β cell number (such as about 10 μgto 1 g, 100 μg to 500 mg, or 1 mg to 100 mg). In other examples, theamount of protein increasing β cell number or a preparation includingthe protein (such as a cell extract or preparation of cells expressingthe protein) to be administered to a subject is about 0.001 mg/kg toabout 1000 mg/kg (such as about 0.01 mg/kg to about 500 mg/kg, about 1mg/kg to about 250 mg/kg, or about 10 mg/kg to 100 mg/kg).

The dosage can be administered one or more times per day, in divideddoses (such as 2, 3, or 4 divided doses per day), or in a single dosagedaily. The dosage can also be administered every 2 days, every 3 days,bi-weekly, once weekly, semi-weekly, or monthly. In some examples, aneffective amount of protein increasing β cell number protein or nucleicacid encoding the protein or a cell-extract or preparation of cellsexpressing the protein is an amount that inhibits or ameliorates one ormore symptoms of diabetes (for example, decreases blood glucose levelsor increases insulin production). In other examples, an effective amountis an amount that increases β cell number in a subject (for example,compared to a control or compared to the subject prior to administrationof the protein increasing β cell number or preparation).

In particular examples, prior to, during, or following administration ofa disclosed protein increasing β cell number (or nucleic acid encodingthe protein, or preparation of cells expressing the protein or cell-freeextract including the protein) the subject can receive one or more othertherapies. Examples of such therapies include, but are not limited to,lifestyle modifications (such as diet and exercise), insulin, metformin,sulfonylureas (such as glyburide, glipizide, or glimepiride),meglitinides (such as repaglinide or nateglinide), thiazolidinediones(such as rosiglitazone or pioglitazone), DPP-4 inhibitors (such assitagliptin, saxagliptin, or linagliptin), GLP-1 receptor agonists (suchas exenatide or liraglutide), and SGLT2 inhibitors (such ascanagliflozin or dapagliflozin). Combinations of one or more of thesetherapies can also be administered to a subject.

V. Methods of Identifying Proteins that Increase β Cell Number

Disclosed herein are methods for identifying modulators of β cell number(such as proliferation, specification, and/or survival), for example,proteins that increase β cell number and/or stimulate β cellproliferation. In some embodiments, the methods include inoculatinggerm-free zebrafish (or a population of germ-free zebrafish) with one ormore defined bacterial strains or CFS supernatant from one or moredefined bacterial strains and/or one or more test compounds anddetermining the number of β cells or β cell proliferation, or a markerof β cell number or proliferation (such as glucose or insulin levels) inthe zebrafish. In some examples, the zebrafish are transgenic for one ormore genes, for example, are transgenic for green fluorescent protein(GFP) or another visibly or otherwise readily detectable proteinexpressed under the control of the insulin promoter. In otherembodiments, the methods include inoculating conventionally raisedzebrafish (or a population of conventionally raised zebrafish) with oneor more test compounds and determining the number or proliferation of βcells or a marker of β cell number or proliferation (such as glucose orinsulin levels).

In some examples, the number of β cells in a fish is determined.Presence or amount of β cells in the pancreas can be determined byhistological staining, in situ hybridization, flow cytometry, orimmunohistochemistry. In one particular example, presence or amount of βcells is determined by detection of a marker expressed under the controlof a neutrophil-specific gene (such as green fluorescent protein (GFP)expressed from the insulin promoter, as described below). In otherexamples, presence or amount of β cells is determined by detection ofone or more proteins expressed by β cells (for example, specificallyexpressed by β cells but not by other pancreatic cells), such asinsulin, diacylglycerol kinase beta (DGKB), or glycoprotein M6A (GPM6A)(see, e.g., Dorrell et al., Mol. Cell. Endocrinol. 339:144-150, 2011).One of ordinary skill in the art can identify additional markers fordetection of presence and/or amount of β cells.

In some examples, the number of β cells or the amount of proliferationof β cells in a zebrafish contacted or treated with a bacterial strain,CFS from a bacterial strain, or other test compound is compared to acontrol. In some examples, the control is a zebrafish (or population ofzebrafish) treated under the same conditions, but without treatment withthe bacterial strain, CFS, or test compound. Bacterial strains or testcompounds identified as modulating β cell number and/or proliferationmay be selected for further testing. If the modulator is a bacterialstrain, additional testing may be carried out to identify or purify oneor more β cell number increasing compounds from the bacteria Bacterialstrains that may be used in the screening methods disclosed herein(either for inoculation of germ-free zebrafish or for preparing CFS withwhich the zebrafish are contacted) include, but are not limited toAeromonas, Shewanella, Photobacterium, Acinetobacter, Pseudomonas,Variovorax, Vibrio, Enterobacter, Plesiomonas, and Delftia. Additionalbacterial strains, such as additional strains found in the zebrafish ormammalian gut (such as the human gut), for example Enterococcus,Klebsiella, Enterobacter, or Escherichia, can also be tested for theability to increase β cell number or stimulate β cell proliferation inthe methods disclosed herein.

A “compound” or “test compound” is any substance or any combination ofsubstances that is useful for achieving an end or result. Any compoundthat has potential (whether or not ultimately realized) to modulate βcell number and/or proliferation can be tested using the methods of thisdisclosure.

Exemplary compounds include, but are not limited to, peptides, such assoluble peptides, including but not limited to members of random peptidelibraries (see, e.g., Lam et al., Nature, 354:82-84, 1991; Houghten etal., Nature, 354:84-86, 1991), and combinatorial chemistry-derivedmolecular libraries made of D- and/or L-configuration amino acids,phosphopeptides (including, but not limited to, members of random orpartially degenerate, directed phosphopeptide libraries; see, e.g.,Songyang et al., Cell, 72:767-778, 1993), antibodies (including, but notlimited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimericor single chain antibodies, and Fab, F(ab′)₂. and Fab expression libraryfragments, and epitope-binding fragments thereof), small organic orinorganic molecules (such as, so-called natural products or members ofchemical combinatorial libraries), molecular complexes (such as proteincomplexes), or nucleic acids (such as antisense compounds).

Appropriate compounds can be contained in libraries, for example,synthetic or natural compounds in a combinatorial library. Numerouslibraries are commercially available or can be readily produced; meansfor random and directed synthesis of a wide variety of organic compoundsand biomolecules, including expression of randomized oligonucleotides,such as antisense oligonucleotides and oligopeptides, also are known.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant and animal extracts are available or can be readilyproduced. Additionally, natural or synthetically produced libraries andcompounds are readily modified through conventional chemical, physicaland biochemical means, and may be used to produce combinatoriallibraries. Such libraries are useful for the screening of a large numberof different compounds.

In some examples, the number of β cells is measured by counting thenumber of β cells in the pancreas (such as the exocrine pancreas) of asubject. Methods for counting β cells include manual counting (forexample examining a sample (such as tissue or an organism under amicroscope) and counting the number of β cells. β cells can beidentified by staining techniques, including histological stains (suchas hematoxylin and eosin) and immunohistochemistry or in situhybridization (for example, using β cell-specific antibodies or probes(such as for insulin, diacylglycerol kinase beta, or glycoprotein M6A)).In other examples, the number of β cells is measured using a label thatis expressed under the control of a neutrophil-specific promoter (suchas transgenic zebrafish expressing green fluorescent protein (GFP) underthe control of the insulin promoter; see, e.g., dilorio et al., Dev.Biol. 244:75-84, 2002). An increase in the number of β cells (such as anincrease of at least about 10%, about 20%, about 50%, about 80%, about90%, about 1.5-fold, about 2-fold, about 3-fold, about 5-fold, about10-fold or more) in mono-associated zebrafish in the presence of one ormore bacterial strains or test compounds as compared to in the absenceof the one or more bacterial strains or test compounds indicates thatthe compound increases β cell number and/or proliferation.

In other examples, β cell proliferation is measured. Methods ofmeasuring cell proliferation are known to one of ordinary skill in theart. Such methods include in vitro or in vivo methods. In some examples,cell proliferation is measured by incorporation of a DNA label (forexample 5-bromo-2-deoxyuridine (BrdU), 5-ethynyl-2′-deoxyuridine, (EdU)or [³H]thymidine). In the presence of label, cells which are in S-phaseincorporate the label. After an incubation period, cells which were inS-phase during the labeling period can be detected, such as byautoradiography (for cells labeled with [³H]thymidine) or withfluorescently-labeled antibodies specific to BrdU (for cells labeledwith BrdU), or appropriate detection reagents (for EdU, such as CLICK-ITEdU kit, Invitrogen). An increase in the number of labeled cells (suchas an increase of about 10%, about 20%, about 50%, about 80%, about 90%,about 1.5-fold, about 2-fold, about 3-fold, about 5-fold, about 10-foldor more) in the presence of one or more bacterial strains, CFS, and/ortest compounds as compared to in the absence of the bacterial strains,CFS, and/or test compounds indicates that the compound increases β cellproliferation.

In other examples, β cell proliferation is measured by detectingcellular DNA content in a population of cells, as DNA content is closelyproportional to cell number. Such methods include detecting a dye thatbinds to nucleic acids (such as CYQUANT cell proliferation kit,Invitrogen). In other examples, cell proliferation is measured byquantifying cleavage of a tetrazolium salt (such as MTT, XTT, or MTS) toinsoluble formazan crystals by mitochondrial dehydrogenase.

In additional examples, β cell proliferation is measured using atransgenic zebrafish line that mark proliferating and quiescent β cells(see, e.g., Tsuji et al., PLoS ONE 9(8):e104112, 2014). One of ordinaryskill in the art can identify additional methods to measurer β cellproliferation.

Glucose levels in zebrafish correlate with the number and/orproliferation of β cells. Therefore, in still further examples, theeffect of a bacterial strain, CFS, and/or test compound on number and/orproliferation of β cells can be determined by measuring glucose levelsin zebrafish treated with the bacterial strains, CFS, and/or testcompounds as compared to zebrafish in the absence of the bacterialstrains, CFS, and/or test compounds.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

Example 1 Materials and Methods

Gnotobiotic zebrafish: All experiments with zebrafish were performedusing protocols approved by the University of Oregon Institutional Careand Use Committee and followed standard protocols. Zebrafish embryoswere derived germ-free (GF) as previously described (Milligan-Myhre etal., Meth. Cell Biol. 105:87-116, 2011). XGF and mono-associated larvaewere also generated as previously described (Bates et al., Dev. Biol.297:374-386, 2006), except that all bacterial inoculums were added tothe GF flasks at 4 days post fertilization (dpf) at a finalconcentration of 10⁶ colony forming units (CFU)/mL. In experimentsquantifying the colonization levels of bacterial isolates, each strainwas added directly to the water and allowed to incubate with the larvaefor 48 hours at 37° C. At 6 dpf the fish were sacrificed before the gutwas removed and homogenized in a small sample of sterile embryo medium(EM). Dilutions of this gut slurry were plated onto tryptic soy agar andallowed to incubate overnight at 30° C. Colonies from each gut werequantified and a minimum of 10 guts per mono-association were analyzed.

Free Glucose Assay: To measure β cell function in GF and CV zebrafishlarvae, levels of free glucose were measured at 6 dpf using a freeglucose assay kit (BioVision) as described previously (Gut et al., Nat.Chem. Biol. 9:97-104, 2013), except that only 10 larvae were combinedper tube. Three to five biological replicates were completed for both GFand CV treatments each time the assay was conducted. Data representedherein have been combined from three separate experimental assays.

Cell Free Supernatant: Secreted bacterial products were inoculated to GFfish at 4 dpf by adding cell free supernatant (CFS) at a finalconcentration of 500 ng/mL to the water of the sterile flasks. CFS washarvested from a 50 mL overnight culture of the specified bacterialstrain. The cultures were centrifuged at 7000×g for 10 minutes at 4° C.Subsequently, the supernatant was filtered through a 0.22-μm steriletube-top filter (Corning Inc., NY). The sterile supernatant wasconcentrated at 4° C. for 1 hour at 3000×g with a centrifugal devicethat has a 10 kDa weight cut off (Pall Life Sciences). For experimentsutilizing proteinase K (Qiagen), the enzyme was added to samples of CFSat a final concentration of 100 μg/mL and allowed to incubate at 55° C.for 1 hour before inactivating the enzyme at 90° C. for 10 minutes.

Ammonium Sulfate Fractionation: Ammonium sulfate fractionation wasperformed on un-concentrated, sterile CFS from a 50 mL overnight cultureby slowly adding 100% ammonium sulfate until solutions of 20%, 40%, 60%or 80% ammonium sulfate were achieved. These solutions were prepared at4° C. Precipitated proteins were collected from each fraction bycentrifugation at 4° C. and 14000 s g for 15 minutes. The proteins wereresuspended in cold embryo medium and dialyzed for 2-3 hours at 4° C.before adding them to GF larvae age 4 dpf at a final concentration of500 ng/mL.

Mass Spectrometry: The 60-80% ammonium sulfate fraction of the A.veronii ^(ΔT2SS) CFS, frozen in liquid nitrogen, were sent to theProteomics Lab at Oregon Health and Sciences University in Portland,Oreg. for protein identification (partial sequencing) analysis.

BefA purification: The nucleotide sequence for the befA gene wasamplified from A. veronii using the following forward and reverse PCRprimers respectively: 5′-GCCCATATGatgaacaagcgtaactggttgctg-3′ (SEQ IDNO: 12) and 5′-GGCCTCGAGgcggctcgtttcagtcaagtc-3′ (SEQ ID NO: 13). Theamplified fragment was then cloned into the pET-21b plasmid (Novagen)which contained an IPTG inducible promoter. A His tag was added to theC-terminal of the protein sequence for subsequent purification. Thisvector was transformed into the BL21 Escherichia coli strain, andtreated with 0.1 mM IPTG during early exponential growth phase(OD₆₀₀=0.4-0.6) and allowed to grow for 3-4 more hours at 30° C. Thisresulted in a supernatant dominated by BefA, as confirmed via SDS-pagegel electrophoresis by the presence of a dark band of the expected sizeof BefA, 29 kDa. This band was absent from BL21 cultures carrying anempty pET-21b vector. IPTG-induced BL21 cells were sonicated at 32,000×gin a 50 nM Tris, 150 mM NaCl buffer (buffer A). The supernatant was thenadded to a solution of nickel beads (Thermo Scientific HisPur™ Ni-NTAResin) to capture the His tag. The beads were washed three times for 15minutes in a 20 mM imidazole solution and subsequently eluted in a 500mM imidazole solution. The isolation of pure BefA was confirmed withSDS-page gel electrophoresis by the presence of a single band of about29 kDa in size (FIG. 4B). Purified BefA was added to 4 dpf GF fish at afinal concentration of 500 ng/mL.

Experimental bacterial strains: To create the A. veronii ^(ΔbefA) mutantstrain, a vector containing a chloramphenicol resistance cassette wastransformed into SM10 E. coli. Conjugation between wild type Aeromonasveronii HM21 and the vector carrying SM10 E. coli strain was carriedout, allowing for the chloramphenicol resistance gene to replace thebefA locus via allelic exchange in A. veronii cells that received thevector. Candidates were selected for loss of the plasmid and maintenanceof chloramphenicol resistance. The insertion of the chloramphenicolcassette into the befA locus was verified in these candidates by PCR.The A. veronii ^(ΔT2SS) strain was provided by Joerg Graf.

β cell quantification and EdU staining: Tg(−1.0insulin:eGFP)¹⁴ zebrafishembryos were used to visualize and quantify the total number of β cellsin the developing larvae. All experiments were analyzed at 6 dpf unlessotherwise specified. For experiments quantifying the proliferation, EdUwas added at 4 dpf directly to the EM at a final concentration of 0.1mg/mL. At all time points and experiments, larvae were fixed with 4%paraformaldehyde at 4° C. overnight and then washed with 1× PBS. TheClick-iT® EdU Imaging Kit (Invitrogen) was used to process the EdU labelin whole fixed zebrafish, except when quantifying IEC proliferation whenfish were sectioned first, according to the manufacturer's protocols.Finally, larvae were also stained with rabbit anti-GFP (LifeTechnologies), Alexa Fluor® 488 goat anti-rabbit (Jackson) andTO-PRO®-3-Iodide (642/661). Whole stained larvae were mounted forconfocal microscopy (BioRad) with their right side facing up against thecover slip, which was flattened sufficiently to disrupt the islet foroptimal resolution of individual cells. For quantification of β cells,the entire endocrine portion of the pancreas was scanned through using a60× objective, and FIJI software was used to analyze each image stack.For quantification of pancreatic exocrine tissue proliferation,Tg(ptf1a:eGFP)³¹ zebrafish were scanned through the entire pancreas witha 20× objective and FIJI was used to analyze the percentage ofproliferative cells in single sections from the center of the organ.

Cell culture: Cell lines, mouse insulinoma 13-TC-6 (ATCC® CRL-11506™)and rat small intestinal epithelia IEC-6 (ATCC® CRL-1592™), werecultured in standard conditions and in DMEM supplemented with 10% FBS.For β-TC-6 cells, low glucose (5 mM) DMEM was used and for the IEC-6cells, medium was additionally supplemented with 0.1 Units/mL of bovineinsulin. Media was changed every 48 hours and cells were split every 7to 9 days. For experiments, cells were split into wells containingpoly-D-lysine coated cover slips. They were allowed to recover for 24hours before adding BefA and EdU at concentrations of 500 ng/mL and 10μM, respectively. Cells were incubated with EdU and BefA or a controlfor 5 hours before the media was removed and the cells were fixed in 4%PFA for 15 minutes at room temperature. Cells were washed three timeswith PBS before developing the EdU signal according to themanufacturer's protocols. Cells were additionally stained with rabbitanti-insulin (Cell Signaling Cat #4590), Alexa Fluor 488 goatanti-rabbit (Jackson) and DAPI before being imaged using a confocalmicroscope (BioRad) with a 20× objective. A minimum of 1,000 β-TC-6cells or 300 IEC-6 cells was quantified per cover slip and the softwareFIJI was used to analyze each image.

BefA phylogenetic analysis: BefA homologs were screened for acrossmicrobial species using a blastp-based search of the UniProtKnowledgebase (version 6/2015) (Altschul et al., Nucl. Acids Res.17:3389-3402, 1997; UniProt Consortium, Nucl. Acids Res. 43:D204-212,2015); default search parameters were changed to allow (i) a maximumE-value of 1.0 and (ii) an arbitrarily large number of database hits.Database hits were classified as “close homologs” if amino acid sequenceidentity exceeded 50% (relative to the query length) and as “distanthomologs” if their percent identity exceeded 20%. For phylogeneticanalysis at the species level, each species was represented by the hitof highest percent identity to BefA among isolates of that species (ifany); an analogous procedure was used for genus-level analysis. Alignedportions of database sequences were isolated and multiply aligned withMUSCLE (Edgar, Nucl. Acids Res. 32:1792-1797, 2004). Phylogenetic treeswere constructed from these multiple sequence alignments using PhyML(Guindon et al., Syst. Biol. 52:696-704, 2003) and visualized within thePhylogeny.fr webserver (Dereeper et al., Nucl. Acids Res. 36:W465-469,2003). Microbial genera were classified as “human-associated” if theyoccurred with relative abundance >0.01% in at least 5 metagenomes fromthe Human Microbiome Project (Human Microbiome Project Consortium,Nature 486:207-214, 2012) as profiled by MetaPhlAn (Segata et al., Nat.Meth. 9:811-814, 2012).

Statistical Analysis: Each experiment was repeated multiple times, anddata was analyzed through the statistical software R Studio. Forexperiments comparing just two differentially treated populations oflarvae, a Student's t-test with equal variance assumptions was used. Forexperiments measuring a single variable (such as total β cells) withmultiple treatment groups, a single factor ANOVA with post hoc meanstesting (Tukey) was utilized. A p-value of less than 0.05 was requiredto reject the null hypothesis that no difference existed between groupsof data.

Example 2 Bacterial Protein that Induces Pancreatic β Cell Proliferation

This example describes identification and characterization of abacterial protein that induces pancreatic β cell proliferation.

By 3 dpf, the larval zebrafish has developed a fully functional pancreaswith a small population of newly differentiated insulin-secreting βcells (Field et al., Dev. Biol. 261:794-208, 2003; Kimmel et al., Meth.Cell Biol. 100:261-280, 2010). During the first week of development,these cells are tightly packed into the larvae's single islet, which canbe readily visualized in transgenic fish expressing eGFP under theinsulin promoter (dilorio et al., Dev. Biol. 244:75-84, 2002). Fromapproximately 3 dpf onward, this population of β cells undergoes alinear expansion, matching the increasing metabolic demands of thegrowing larvae (Tsuji et al., PLoS One 9:e104112, 2014; Moro et al.,Dev. Biol. 332:299-308, 2009). Both self-proliferation and neogenesiscontribute to the growing β cell mass in the zebrafish (Hesselson etal., Proc. Natl. Acad. Sci. USA 106:14896-14901, 2009). At the outset ofthis expansion period, between 3 and 4 dpf, bacteria from theenvironment colonize the zebrafish intestine for the first time (Bateset al., Dev. Biol. 297:374-386, 2006). Human infants undergo a similar βcell expansion event, characterized by increased levels of proliferation(Gregg et al., J. Clin. Endocrinol. Metab. 97:3197-3206, 2012), whichalso occurs during initial establishment of the gut microbiota (Voreadeset al., Front. Microbiol. 5:494, 2014).

To investigate a possible role for the microbiota in β cell expansion,total eGFP⁺ cells in germ free (GF) and conventionally reared (CV)Tg(−1.0insulin:eGFP) fish (dilorio et al., Dev. Biol. 244:75-84, 2002)at 3, 4, 5 and 6 dpf were quantified. The number of β cells in CV fishincreased steadily from 3 to 6 dpf; however, the number of β cells in GFfish remained stagnant over this time (FIG. 1A). Furthermore, at 6 dpf,the overall structure of β cells within the primary islet of CV fish(FIG. 1B) appeared different from that of GF fish (FIG. 1C), with cellsin the GF animals being less densely packed. β cell numbers were rescuedto CV levels by the addition of non-sterile tank water to GF larvae by 4dpf (FIG. 1D, XGF). These results suggested that development of acomplete β cell population was dependent upon microbes ormicrobial-derived products.

In order to determine whether this microbe-dependent β cell deficiencycould affect the metabolic function of the fish, the levels of glucosepresent in both GF and CV larvae at 6 dpf were measured. The amount ofglucose detected in GF fish was significantly higher than in CV fish(FIG. 1E), suggesting that GF fish are less efficient at importing andprocessing glucose from the blood, consistent with lower levels ofcirculating insulin in GF larvae with a paucity of β cells.

To investigate the possibility that a single bacterial species canpromote β cell expansion, β cell numbers in larvae monoassociated at 4dpf with bacterial isolates of the zebrafish intestinal microbiota thathave been shown to colonize the larval gut in isolation were measured(FIG. 2). Three different species of the genus Aeromonas, and onespecies of the genus Shewanella were each sufficient to rescue GF β cellnumbers to levels observed in CV fish (FIG. 1D), supporting theconclusion that specific members of the microbiota are capable ofinducing expansion of the β cell mass.

It is well documented that bacterial interactions with host organismsoften involve secreted molecules or proteins. Therefore, to test whethera secreted bacterial factor(s) could influence β cell expansion, cellfree supernatant (CFS) was harvested from overnight cultures ofAeromonas veronii HM21 (A. veronii), one of the species shown to rescueβ cell expansion (FIG. 1D). The CFS from A. veronii cultures was addedto GF larvae at 4 dpf. The CFS alone was able to restore GF β cellnumbers (FIG. 3A), indicating that a secreted factor (or factors) wassufficient to induce normal β cell expansion. As a control, GF fish weretreated with CFS from a Vibrio sp. isolate, which colonized thezebrafish gut (FIG. 2, ★), but did not induce β cell expansion (FIG. 1D,★). This treatment was not significantly different from GF (FIG. 3A).Furthermore, the capacity to induce β cell numbers was lost when the A.veronii CFS sample was treated with proteinase K (FIG. 3A), indicatingthat the secreted factor(s) of interest was highly likely to be aprotein.

In order to narrow down the list of candidates secreted by A. veronii,activity in the CFS of an A. veronii ^(ΔT2SS) mutant strain lacking afunctional type 2 secretion system (T2SS; Maltz et al., Appl. EnvironMicrobiol. 77:597-603, 2011), the major protein secretion pathway ofGram-negative bacteria (Cianciotto, Trends Microbiol. 13:581-588, 2005),was tested. Surprisingly, CFS harvested from the A. veronii ^(ΔT2SS)strain was still sufficient to rescue GF β cell numbers (FIG. 3A).Ammonium sulfate precipitation was used to further separate proteinswithin the A. veronii ^(ΔT2SS) CFS. One of these fractions was able toinduce β cell numbers equivalent to those found in CV fish (FIG. 4A,60-80% ammonium sulfate). Mass spectrometry was used to analyze thecontent of this fraction, which led to the identification of 187distinct proteins (Table 1).

TABLE 1 Proteins in active fraction identified by mass spectrometrySpectral counts Protein description 188 Peptide ABC transportersubstrate-binding protein 125 Enolase 91 Elongation factor Ts 87Uncharacterized protein 74 Dihydrolipoyl dehydrogenase 71 50S ribosomalprotein L9 70 Phosphoenolpyruvate carboxykinase [ATP] 69Glyceraldehyde-3-phosphate dehydrogenase 62 Membrane protein 54 Malatedehydrogenase 54 Azurin 49 Cytochrome C 44 Transaldolase 41Triosephosphate isomerase 40 50S ribosomal protein L1 40Fructose-bisphosphate aldolase 37 Putrescine-binding periplasmic protein37 N utilization substance protein B homolog 33 Phosphoglycerate kinase29 50S ribosomal protein L19 29 50S ribosomal protein L7/L12 23C4-dicarboxylate ABC transporter substrate-binding protein 22 Sugar ABCtransporter substrate-binding protein 21 LafB (Fragment) 21 Transporter20 Uncharacterized protein 20 Universal stress protein 19 10 kDachaperonin 19 Superoxide dismutase 182,3-bisphosphoglycerate-independent phosphoglycerate mutase 18Uncharacterized protein 16 Transcriptional regulator 15 Chaperoneprotein DnaK 15 Probable thiol peroxidase 15 50S ribosomal protein L1014 30S ribosomal protein S30 14 Thioredoxin 14 Uncharacterized protein13 Serine protease 13 30S ribosomal protein S16 13 RNApolymerase-binding transcription factor DksA 13 Amino acid ABCtransporter substrate-binding protein 12 50S ribosomal protein L24 12Translation initiation factor IF-2 11 50S ribosomal protein L6 11Arginine ABC transporter substrate-binding protein 11 D-ribosetransporter subunit RbsB 11 Elongation factor P 11 RNA-binding protein11 Glutamine-tRNA ligase 11 Chemotaxis protein CheY 10 Peptidase M16 1050S ribosomal protein L22 10 UPF0234 protein 10 Succinyldiaminopimelateaminotransferase 10 Peptidyl-prolyl cis-trans isomerase 10Transcriptional regulator 9 Fumarate hydratase 9 RNAchaperone/anti-terminator 9 Peptide ABC transporter substrate-bindingprotein 9 Methyl-galactoside ABC transporter substrate-binding protein 9Methionine aminopeptidase 9 30S ribosomal protein S6 9 Ornithinecarbamoyltransferase 9 Ribosome-recycling factor 8 Peptidyl-prolylcis-trans isomerase 8 Uncharacterized protein 8 Uncharacterized protein8 30S ribosomal protein S1 7 Anti-sigma D factor 7 Dihydrolipoamidesuccinyltransferase 7 Malonyl CoA-acyl carrier protein transacylase 7Organic solvent ABC transporter substrate-binding protein 6Phosphoribosylaminoimidazole-succinocarboxamide synthase 6 Acetatekinase 6 Uncharacterized protein 6 Cell wall assembly/cell proliferationcoordinating protein 6 Elongation factor Tu (Fragment) 6 Iron ABCtransporter substrate-binding protein 6 Deoxyribose-phosphate aldolase 6Transcription elongation factor GreA 6 50S ribosomal protein L25 6Tungsten ABC transporter substrate-binding protein 6 Cyclic diguanosinemonophosphate-binding protein 6 Chaperone protein HtpG 6 RNAchaperone/anti-terminator 6 ABC transporter substrate-binding protein 6Peptidase M54 5 Thiosulfate sulfurtransferase 5 Isocitrate lyase 5 Lonprotease 5 3-hydroxydecanoyl-[acyl-carrier-protein] dehydratase 5Glutamate--cysteine ligase 5 Amino acid ABC transportersubstrate-binding protein 5 Phosphopentomutase 5 Peptidyl-prolylcis-trans isomerase 5 Transcriptional regulator 4 DNA-directed RNApolymerase subunit alpha 4 Glutamate--tRNA ligase 4 Uncharacterizedprotein 4 30S ribosomal protein S2 4 Signal recognition particlereceptor FtsY 4 2′,3′-cyclic nucleotide 2′-phosphodiesterase 4 Regulatorof ribonuclease activity A 4 30S ribosomal protein S17 4 Exonuclease III4 Uncharacterized protein 4 Acetylornithine aminotransferase 4Endoribonuclease L-PSP 4 Cold-shock protein 4 Trigger factor 4Glutamine--fructose-6-phosphate aminotransferase [isomerizing] 4Methionine--tRNA ligase 4 Lysine--tRNA ligase 4 Uncharacterized protein4 PTS glucose transporter subunit IIA 4 L-asparaginase 4 Anti-RNApolymerase sigma 70 factor 4 Elongation factor G 3 Uncharacterizedprotein 3 Phosphate-binding protein 3 Single-stranded DNA-bindingprotein 3 UDP-N-acetylglucosamine 1-carboxyvinyltransferase 3Glutaredoxin 3 Preprotein translocase subunit Tim44 3 30S ribosomalprotein S13 3 6,7-dimethyl-8-ribityllumazine synthase 3 Oxidoreductase 350S ribosomal protein L11 3 DNA-directed RNA polymerase subunit beta′ 3Translation initiation factor IF-I 3 Amino acid ABC transportersubstrate-binding protein 3 Polyribonucleotide nucleotidyltransferase 3Heme ABC transporter ATP-binding protein 2 Exodeoxyribonuclease Vsubunit gamma 2 Thiol:disulfide interchange protein 2 Uncharacterizedprotein 2 Peptidyl-tRNA hydrolase 2 Putative agmatine deiminase 2Peptidoglycan-binding protein 2 Membrane protein 2 Outer membraneprotein assembly factor BamC 2 Probable endonuclease 4 2 Uncharacterizedprotein 2 Glucosaminidase 2 ABC transporter substrate-binding protein 21-(5-phosphoribosyl)-5-[(5- phosphoribosylamino)methylideneamino]imidazole-4-carboxamide isomerase 2 PTS fructose transporter subunit IIC2 Amidase 2 Molybdate ABC transporter substrate-binding protein 2Membrane protein 2 Zn-dependent protease 2 Uncharacterized protein 2Uncharacterized protein 2 Phosphoserine aminotransferase 2 Riboflavinsynthase subunit alpha 2 Putrescine-binding periplasmic protein 2Aldo/keto reductase 2 Uncharacterized protein 2 Chemotaxis protein CheW2 Lipoprotein 2 Uncharacterized protein 2 Membrane protein

To identify promising candidates from this list, the fact thatzebrafish-associated bacterial isolates have differential abilities toinduce larval β cells (FIG. 1D) was utilized. Basic local alignmentsearch tool (BLAST) was used to identify those candidate proteinsencoded by the genomes of bacterial strains that were sufficient torestore host β cells, and absent from those strains which were notsufficient. This approach identified a single gene, denoted by the locustag, M0001_10165, predicted to encode a putative protein of 261 aminoacids (protein highlighted in Table 1). The relative abundance of thiscandidate was low compared to other species detected within the CFSfraction, suggesting that it is a rare product of the bacterialmicrobiota.

In order to test whether M001_10165 encoded the protein responsible forinducing β cell numbers, it was cloned it into an expression vector withan added C-terminal His tag for subsequent purification from BL21Escherichia coli (FIG. 4B). Addition of purified M001_10165 (10165)protein to GF zebrafish larvae was sufficient to rescue β cell numbers(FIG. 3B). Image comparisons of the islets from differentially treatedfish were striking, as both the CV and GF β cell populations weredwarfed by the expanded islets of several of the fish treated with 10165(FIGS. 3C-E). Therefore, this protein has been named β cell ExpansionFactor A (BefA), after its observed function in the zebrafish.

To determine whether the befA locus was necessary to induce an increasein β cell numbers in fish mono-associated with A. veronii, an A. veronii^(ΔbefA) mutant strain was generated by replacing the coding region ofbefA with a chloramphenicol resistance gene. To ensure that the loss ofthe befA gene would not affect the ability of A. veronii to form amono-association with the larvae, colonization assays were performed nodeficiency in the ability of A. veronii ^(ΔBefA) to colonize the gutcompared to A. veronii ^(WT) was observed (FIG. 2). GF fish weremono-associated with the A. veronii ^(ΔbefA) strain, or treated with itsCFS from 4 to 6 dpf. Neither treatment was sufficient to rescue β cellnumbers to CV totals (FIG. 3B). However, mono-associations of A. veronii^(ΔbefA) could be complemented in trans with CFS from A. veronii ^(WT),which resulted in complete restoration of the β cell population (FIG.3B). Taken together, these data demonstrate that a single protein,produced by a specific subset of bacteria within the zebrafishmicrobiota, is both necessary and sufficient for early β cell massexpansion.

To understand how BefA induces zebrafish β cell expansion, whether BefAcould increase β cell proliferation was tested. GF larvae were treatedwith BefA from 4 to 6 dpf in the presence of the thymidine analog,5-ethynyl-2′-deoxyuridine (EdU). β cells of both BefA treated and CVfish exhibited significantly elevated levels of proliferation comparedto GF fish (FIG. 5A-G). However, CFS from the A. veronii ^(ΔbefA) strainwas not sufficient to increase proliferation rates in GF fish (FIG. 5G).These results suggest that BefA is both necessary and sufficient toincrease basal rates of β cell proliferation during early larvaldevelopment. This is the first demonstration that a bacterial-basedproduct can cause β cell proliferation. Other mechanisms, such asneogenesis (Wang et al., Development 138:609-617, 2011) andtransdifferentiation from alpha cells (Ye et al., Development142:1407-1417, 2015), have been shown to contribute to the β cellpopulation, and may also be influenced by BefA.

BefA was also tested for its effect on proliferation rates of mammalianβ cells. BefA was added to murine 13-TC-6 cells (Poitout et al.,Diabetes 44:306-313, 1995) in the presence of EdU (FIG. 5I). In samplestreated with BefA, a significantly greater percentage of insulinexpressing cells underwent cell division than in samples that did notreceive the BefA treatment (FIG. 5H), showing that BefA, a bacterialproduct isolated from the zebrafish microbiota, is sufficient toincrease proliferation in mammalian β cells.

To gain insight into the specificity of BefA in host tissues, itsability to induce proliferation in both the exocrine pancreas and in theintestinal epithelium of zebrafish larvae was also tested. The levels ofproliferation in these tissues were not changed across GF, CV and BefAtreated larvae (FIGS. 6A and B), suggesting that BefA acts in atissue-specific manner. Furthermore, BefA had no effect on levels ofproliferation in the murine intestinal epithelial cell line IEC-6 (FIG.6C), suggesting that BefA acts upon pancreatic endocrine tissue frommultiple species.

Although befA is not required for the formation of mono-associations, itcould be important to bacterial fitness by playing a role as acolonization factor. Colonization factor genes are often important forcompetition within the host environment. Therefore to investigate apotential role for befA in bacterial competition, GF fish wereco-inoculated with equal concentrations of A. veronii ^(WT) and A.veronii ^(ΔbefA). Interestingly, after allowing 48 hours forcolonization to occur, the A. veronii ^(ΔbefA) strain was outcompeted inthe gut by A. veronii ^(WT) (FIG. 2B), suggesting that befA could playan important fitness role for A. veronii as a colonization factor gene.

Phylogenetic analysis of BefA revealed a close homolog (82% amino acidsequence identity) in the human-associated species Enterococcusgallinarum, along with homologs in many species of the Aeromonas,Vibrio, and Photobacterium genera (FIG. 7A). Widening the search toinclude more distant homologs identified potentially related genes inthree additional human-associated genera: Enterobacter, Escherichia, andKlebsiella (FIG. 7B). These results suggest that a similar host-microbesignaling mechanism could exist within humans. Additionally, the BefAamino acid sequence contains a putative SYLF domain in the C-terminal,and this domain is also predicted within each human-associated homologof BefA. Interestingly, the domain is conserved across the kingdom oflife and has lipid-binding function in more complex organisms (Hasegawaet al., J. Cell Biol. 193:901-906, 2011).

Example 3 Methods of Screening for Modulators of β Cell Number

This example describes particular methods that can be used for screeningfor modulators of β cell number utilizing transgenic zebrafish thatexpress GFP under the control of the insulin promoter. One skilled inthe art will appreciate that methods that deviate from these specificmethods can also be used to successfully screen for modulators of β cellnumber.

Day 1: Set up Ins: GFP fish to cross naturally. Use dividers to preventegg laying until the morning.

Day 2 (0 dpf):

-   -   1. By 9:00 am, move natural crosses into tanks with fresh water        and pull dividers to allow fish to mate. Prepare antibiotic EM        to collect eggs (100 μg/mL ampicillin, 5 μg/mL kanamycin, and        250 μg/ml amphotericin B, sterile filtered). Collect eggs in        antibiotic EM and place in 30° C. incubator until they reach        shield stage.    -   2. Move embryos into sterile 50-mL beaker. Wash embryos 3× in        sterile EM. Immerse embryos in 0.1% PVP-I solution for 2        minutes. Rinse 3× in sterile EM. Transfer embryos to a new        sterile 50-mL beaker and immerse in 0.003% bleach for 20        minutes. Pour off bleach and rinse 3× in sterile EM. Transfer        20-25 embryos into sterile cell culture flasks with 50-mL        sterile EM.

Day 5 (3 dpf):

-   -   1. Start overnight bacterial cultures. Start 50-mL cultures of        bacteria for testing CFS for beta cell proliferation activity.

Day 6 (4 dpf):

-   -   1. Follow CFS preparation protocol listed above (Example 2).    -   2. Visually check GF fish flasks for bacteria.    -   3. Inoculate flasks with 10⁶ cfu/ml bacteria of interest or with        500 ng/ml concentrated CFS of interest. For each experiment,        include one CV flask as a control and a GF flask as a control.        Collect 1-mL of flask water before inoculation to plate and        confirm that flasks were germ free at the start of the        experiment.

Day 8 (6 dpf):

-   -   1. Check the plates with the inoculation water to ensure the        flasks were germ-free before the experiment started.    -   2. One flask at a time, add tricaine to the fish to anesthetize        them. Use a glass pipette to carefully pull each fish out of the        flask and place it into a 1.5 mL tube. Put up to 20 fish of the        same treatment into a single tube. Remove all excess EM from the        tube and add 1 mL of 4% PFA in PBS with 0.1% Triton X. Place        tubes at 4° C. with gentle rocking overnight.

Day 9: Processing for imaging

-   -   1. If detecting EdU, follow protocol above according to the        manufacturer's protocol. Each tube of fish can be treated as one        “cover slip” in the protocol.    -   2. Add TO-PRO® nuclear stain as described above in 2% BSA and        leave at 4° C. overnight with gentle rocking.    -   3. Wash each tube 8 times for at least 15 minutes at room        temperature with 1 mL of PBS. Mount whole fish onto a glass        slide. Orient the fish so that they are lying with the right        side of the body facing up to most easily visualize the        pancreatic islet.    -   4. Use confocal microscopy to scan the entire islet using a 60×        objective with a Z-stack size of roughly 20 μM, and a step size        of 0.2 μM to ensure good resolution of all the beta cells in the        islet.

Quantify beta cells by counting the total number of nuclei that expressGFP.

Example 4 Method of Determining Effect of BefA Proteins on β Cells in aMammalian Subject

This example describes particular methods that can be used to determinethe effect of BefA or related proteins on β cell number or proliferationin a mammalian subject. One skilled in the art will appreciate thatmethods that deviate from these specific methods can also besuccessfully used.

In order to determine whether BefA (such as SEQ ID NOs: 1-7 disclosedherein) or a related protein can increase the β cell mass in mice, atransgenic mouse model with the insulin promoter driving expression of afluorescent marker (such as GFP) or a mouse model of diabetes (such asthe NOD mouse model) is utilized. Purified BefA protein or a bacterialstrain common to the mouse intestinal microbiota, such as E. coli,engineered to overproduce and secrete BefA is administered (for example,via oral gavage into the gastrointestinal tract) according to standardprotocols. Adult and newborn mice can be tested. After several days(e.g., 1-7 days), mice are sacrificed and the β cell mass is quantifiedaccording to histology techniques and compared to mice treated withappropriate controls. β cell proliferation is analyzed by processinghistological sections with EdU. In diabetes model mice, the incidenceand/or severity of disease symptoms is also monitored. An increase in βcell mass and/or β cell proliferation compared to control mice indicatesthat the protein increases β cell number. A decrease in the incidence ofdiabetes and/or in the number or severity of symptoms in diabetes modelmice also indicates that the protein increases β cell number and/or canbe used to treat or inhibit diabetes.

Example 5 Method of Treating or Inhibiting Diabetes

This example describes particular methods that can be used to treat orinhibit diabetes and/or increase β cell number or proliferation in asubject. However, one skilled in the art will appreciate that methodsthat deviate from these specific methods can also be successfully used.

Based upon the teaching disclosed herein, β cell number can be increasedand diabetes can be treated or inhibited by administering an effectiveamount of a composition including an Aeromonas protein increasing β cellnumber, a nucleic acid encoding the protein, or a preparation includingcells that produce the protein or a cell-free supernatant from suchcells to a subject with diabetes.

In an example, a subject with diabetes (such as a subject with Type Idiabetes) is identified and selected for treatment. Following subjectselection, an effective dose of the composition or preparation includingthe protein increasing β cell number, nucleic acid, or cells orcell-free supernatant described above is administered to the subject.The amount of the composition or preparation administered to prevent,reduce, inhibit, and/or treat diabetes depends on the subject beingtreated, the severity of the disorder, and the manner of administrationof the composition. Ideally, an effective amount of an agent is theamount sufficient to prevent, reduce, and/or inhibit, and/or treat thecondition (e.g., diabetes) in a subject without causing substantialadverse effects in the subject.

In one specific example, a protein increasing β cell number (such as aprotein comprising, consisting essentially of, or consisting of thesequence of SEQ ID NOs: 1-7), a fragment thereof (such as a SYLF domain,for example, amino acids 114-258 of any one of SEQ ID NOs: 1-3), or acell expressing the protein or fragment thereof, is administered to asubject. For example, a protein increasing β cell number is administeredto a subject at about 1 mg to 1 g daily. In another example, a proteinincreasing β cell number is administered at about 1 mg to 1 g biweeklyor weekly. In further examples, a nucleic acid encoding a proteinincreasing β cell number (such as SEQ ID NOs: 8-11) is administered to asubject at about 1 mg to 1 g daily, biweekly, or weekly. An appropriatedose can be selected by a skilled clinician based on the subject, thecondition being treated and other factors.

Subjects are monitored by methods known to those skilled in the art todetermine responsiveness of the subject to the treatment. For example,the symptoms of the subject are monitored, for example blood glucoselevels, blood insulin levels, insulin sensitivity index, homeostaticmodel assessment score, quantitative insulin sensitivity check indexscore (QUICKI; Katz et al., J. Clin. Endocrinol. Metab. 85:2402-2410,2000), or a combination of two or more thereof. It is contemplated thatadditional agents can be administered, such as additional diabetestherapeutics in combination with or following treatment with theAeromonas protein increasing β cell number.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention. Rather, the scope of the invention is defined bythe following claims. We therefore claim as our invention all that comeswithin the scope and spirit of these claims.

We claim:
 1. A method for treating or inhibiting diabetes in a subject,comprising administering to the subject an effective amount of: apolypeptide comprising an amino acid sequence having at least 95%sequence identity to the amino acid sequence of any one of SEQ ID NOs:1-7, thereby treating or inhibiting diabetes in the subject.
 2. Themethod of claim 1, wherein treating or inhibiting diabetes comprisesincreasing β cell number and/or proliferation in the subject.
 3. Themethod of claim 1, wherein the effective amount of the polypeptideadministered to the subject is about 1 mg to about 5 g.
 4. The method ofclaim 1, wherein the effective amount of the polypeptide is administeredto the subject daily, weekly, or monthly.
 5. The method of claim 1,further comprising administering one or more additional diabetestherapies to the subject.
 6. A method for increasing β cell number orproliferation, comprising contacting pancreatic cells with an effectiveamount of: a polypeptide comprising an amino acid sequence having atleast 95% sequence identity to the amino acid sequence of any one of SEQID NOs: 1-7, thereby increasing β cell number or proliferation.
 7. Themethod of claim 6, wherein contacting pancreatic cells comprisesadministering the polypeptide to a subject.
 8. The method of claim 7,wherein the subject has diabetes.
 9. The method of claim 7, wherein thesubject is a fish.
 10. The method of claim 8, further comprisingadministering one or more additional diabetes therapies to the subject.11. The method of claim 1, wherein the polypeptide comprises the aminoacid sequence of any one of SEQ ID NOs: 1-7.
 12. The method of claim 11,wherein the polypeptide consists of the amino acid sequence of any oneof SEQ ID NOs: 1-7.
 13. The method of claim 11, wherein the polypeptideis encoded by a nucleic acid comprising the sequence of any one of SEQID NOs: 8-11.
 14. The method of claim 6, wherein the polypeptidecomprises the amino acid sequence of any one of SEQ ID NOs: 1-7.
 15. Themethod of claim 14, wherein the polypeptide consists of the amino acidsequence of any one of SEQ ID NOs: 1-7.
 16. The method of claim 14,wherein the polypeptide is encoded by a nucleic acid comprising thesequence of any one of SEQ ID NOs: 8-11.